Compositions and methods for the treatment of sepsis

ABSTRACT

The present invention relates to compositions and methods for the prevention and treatment of blood-borne and toxin mediated diseases, and in particular anti-C5a antibodies for the prevention and treatment of sepsis in humans as well as other animals. The present invention also relates to methods of generating anti-C5a antibodies employing C-terminal truncated C5a peptides.

This application is a continuation of copending U.S. patent applicationSer. No. 09/387,671, filed Aug. 31. 1999.

This invention was made with Government support under the NationalInstitutes of Health (NIH) awarded by contract GM29507 and HL31963 Thegovernment has certain rights in this invention.

FIELD OF THE INVENTION

The present invention relates to compositions and methods for theprevention and treatment of blood-borne and toxin-mediated diseases, andin particular anti-C5a antibodies for the prevention and treatment ofsepsis in humans as well as other animals.

BACKGROUND OF THE INVENTION

Sepsis is a major cause of morbidity and mortality in humans and otheranimals. It is estimated that 400,000-500,000 episodes of sepsisresulted in 100,000-175,000 human deaths in the U.S. alone in 1991.Sepsis has become the leading cause of death in intensive care unitsamong patients with non-traumatic illnesses. [G. W. Machiedo et al.,Surg. Gyn. & Obstet. 152:757-759 (1981).] It is also the leading causeof death in young livestock, affecting 7.5-29% of neonatal calves [D. D.Morris et al., Am. J Vet. Res. 47:2554-2565 (1986)], and is a commonmedical problem in neonatal foals. [A. M. Hoffman et al., J. Vet. Int.Med. 6:89-95 (1992).] Despite the major advances of the past severaldecades in the treatment of serious infections, the incidence andmortality due to sepsis continues to rise. [S. M. Wólff, New Eng. J.Med. 324:486-488 (1991).]

Sepsis is a systemic reaction characterized by arterial hypotension,metabolic acidosis, decreased systemic vascular resistance, tachypneaand organ dysfunction. Sepsis can result from septicemia (i.e.,organisms, their metabolic end-products or toxins in the blood stream),including bacteremia (i.e., bacteria in the blood), as well as toxemia(i.e., toxins in the blood), including endotoxemia (i.e., endotoxin inthe blood). The term “bacteremia” includes occult bacteremia observed inyoung febrile children with no apparent foci of infection. The term“sepsis” also encompasses fungemia (i.e., fungi in the blood), viremia(i.e., viruses or virus particles in the blood), and parasitemia (i.e.,helminthic or protozoan parasites in the blood). Thus, septicemia andseptic shock (acute circulatory failure resulting from septicemia oftenassociated with multiple organ failure and a high mortality rate) may becaused by a number of organisms.

The systemic invasion of microorganisms presents two distinct problems.First, the growth of the microorganisms can directly damage tissues,organs, and vascular function. Second, toxic components of themicroorganisms can lead to rapid systemic inflammatory responses thatcan quickly damage vital organs and lead to circulatory collapse (i.e.,septic shock) and oftentimes, death.

There are three major types of sepsis characterized by the type ofinfecting organism. Gram-negative sepsis is the most common and has acase fatality rate of about 35%. The majority of these infections arecaused by Escherichia coli, Klebsiella pneumoniae and Pseudomonasaeruginosa. Gram-positive pathogens such as the Staphylococci andStreptococci are the second major cause of sepsis. The third major groupincludes fungi, with fungal infections causing a relatively smallpercentage of sepsis cases, but with a high mortality rate.

Many of these infections are acquired in a hospital setting and canresult from certain types of surgery (e.g., abdominal procedures),immune suppression due to cancer or transplantation therapy, immunedeficiency diseases, and exposure through intravenous catheters. Sepsisis also commonly caused by trauma, difficult newborn deliveries, andintestinal torsion (especially in dogs and horses).

Many patients with septicemia or suspected septicemia exhibit a rapiddecline over a 24-48 hour period. Thus, rapid methods of diagnosis andtreatment delivery are essential for effective patient care.Unfortunately, a confirmed diagnosis as to the type of infectiontraditionally requires microbiological analysis involving inoculation ofblood cultures, incubation for 18-24 hours, plating the causativeorganism on solid media, another incubation period, and finalidentification 1-2 days later. Therefore, therapy must be initiatedwithout any knowledge of the type and species of the pathogen, and withno means of knowing the extent of the infection.

It is widely believed that anti-endotoxin antibody treatmentadministered after sepsis is established may yield little benefitbecause these antibodies cannot reverse the inflammatory cascadeinitiated by endotoxin. In addition, the high cost of each antibodycould limit physicians' use of a product where no clear benefit has beendemonstrated. [K. A. Schulman et al., JAMA 266:3466-3471 (1991).]Furthermore, these endotoxin antibodies only target gram-negativesepsis, and no equivalent antibodies exist for the array ofgram-positive organisms and fungi.

Clearly, there is a great need for agents capable of preventing andtreating sepsis. It would be desirable if such agents could beadministered in a cost-effective fashion. Furthermore, approaches areneeded to combat all forms of sepsis.

SUMMARY OF THE INVENTION

The present invention relates to compositions and methods for theprevention and treatment of blood-borne and toxin mediated diseases, andin particular anti-C5a antibodies for the prevention and treatment ofsepsis in humans as well as other animals.

The present invention provides a composition comprising antibodyspecific for complement component C5a peptide. In another embodiment,the composition comprises antibody which is specific for complementcomponent C5a peptide, wherein the C5a peptide has a C-terminal regionand an N-terminal region, and the antibody is not reactive with theC-terminal region. In further embodiments, the antibody is specific forthe N-terminal region of complement component C5a peptide. In anadditional embodiment, the antibody is also not reactive with complementcomponent C5 protein.

It is not intended that the present invention be limited to antibodiesspecific for C5a peptides from certain animals. In certain embodiments,the antibody is specific for rat C5a peptide. In other embodiments, theantibody is specific for bovine C5a peptide. In still other embodiments,the antibody is specific for porcine C5a peptide. In a preferredembodiment, the antibody is specific for human C5a peptide.

It is also not intended that the present invention be limited toantibodies generated in a particular animal. A variety of animals areuseful for generating the antibodies of the present invention. In oneembodiment, the antibody is generated in an animal selected from amouse, a rat, a horse, a goat, a chicken, and a rabbit. In someembodiments, the antibodies are collected from the blood of the animal.In other embodiments, the animal generating the antibodies is a bird,and the antibodies are collected from egg yolk.

It is not intended that the present invention be limited to the natureof the antibodies, as a variety of antibody types are contemplated. Inone embodiment, the antibodies are monoclonal. In another embodiment,the antibodies are humanized. In other embodiments, the antibodies arechimaeric. In a preferred embodiment, the antibodies are polyclonal.

The present invention also provides a method of producing polyclonalantibody. In one embodiment, the method comprises, providing; an animaland an immunogenic composition, wherein the composition comprisesC-terminal truncated C5a peptides; and immunizing the animal with theimmunogenic composition in order to generate antibodies. In someembodiments, the immunogenic composition comprises adjuvant. In afurther embodiment, antibodies are collected from the animal.

It is not intended that the present invention be limited to antibodiesspecific for C5a peptides from any particular animal. In certainembodiments, the antibody is specific for rat C5a peptide. In otherembodiments, the antibody is specific for bovine C5a peptide. In stillother embodiments, the antibody is specific for porcine C5a peptide. Ina preferred embodiment, the antibody is specific for human C5a peptide.

It is not intended that the present invention be limited to particularC-terminal truncated peptides. A variety of C-terminal truncatedpeptides are contemplated. In one embodiment, the C-terminal truncatedpeptide corresponds to the entire N-terminal region of C5a peptide. Inanother embodiment, the C-terminal truncated peptide corresponds to theentire N-terminal region of C5a peptide and a portion of the C-terminalregion. In another embodiment, the C-terminal truncated peptide is afragment or portion of the N-terminal region of C5a peptide. In anotherembodiment, the C-terminal truncated C5a peptide is betweenapproximately 5 and 50 amino acids in length. In some embodiments, theC-terminal truncated peptide is approximately fifty amino acids inlength. In other embodiments, the C-terminal truncated peptide isapproximately five amino acids in length. In preferred embodiments, theC-terminal truncated peptides are 20 amino acids in length. In certainembodiments, the C-terminal truncated peptides are selected from SEQ IDNOS:2, 4, 5, 14, 15, and 16.

The present invention also provides a method of treating a subject withthe antibodies of the present invention. In one embodiment, the methodcomprises; providing; a subject, and a therapeutic compositioncomprising an antibody specific for complement component C5a peptide,wherein the C5a peptide has a C-terminal region and an N-terminalregion, and wherein the antibody is not reactive with the C-terminalregion; and administering the therapeutic composition to the subject. Inanother embodiment, the antibody is specific for the N-terminal regionof complement component C5a peptide.

In one embodiment, the present invention provides a method comprising;providing; a subject, and a therapeutic composition comprising anantibody specific for complement component C5a peptide, wherein the C5apeptide has a C-terminal region and an N-terminal region, and whereinthe antibody is not reactive with the C-terminal region; andadministering the therapeutic composition to the subject. In anotherembodiment, administering the therapeutic composition reduces thebinding of complement component C5a peptide to one or more neutrophilsof the subject. In a certain embodiment, administering the therapeuticcomposition reduces bacteremia in the subject. In yet anotherembodiment, administering the therapeutic composition increases the H₂O₂production of neutrophils of the subject. In a preferred embodiment,administering the therapeutic composition reduces the symptoms ofsepsis.

It is not intended that the therapeutic method of the present inventionbe limited to particular subjects. A variety of subjects arecontemplated. In one embodiment the subject is selected from a pig, arat, a cow, a horse, and a human. In one embodiment, the therapeuticcomposition is administered to a subject suffering from symptoms ofsepsis. In another embodiment, the therapeutic composition isadministered prophylactically to a subject at risk for sepsis, includingnew born humans and animals.

It is not intended that the therapeutic method of the present inventionbe limited to certain modes of administration. A variety of modes ofadministering the therapeutic composition are contemplated. In oneembodiment, the therapeutic composition is administered by a modeselected from intravenously, intramuscularly, subcutaneously,intradermally, intraperitoneally, intrapleurally, intrathecally, andtopically.

It is not intended that the present invention be limited to a particulartherapeutic composition. A variety of compositions are contemplated. Inone embodiment the therapeutic composition comprises a soluble mixtureof anti-C5a antibodies. In another embodiment, the anti-C5a antibodiesare provided together with physiologically tolerable liquid, gels, solidcarriers, diluents, adjuvants or excipients, and combinations thereof.In other embodiments, the therapeutic composition comprises anti-C5aantibodies and other therapeutic agents (e.g. other immunoglobulins orantibiotics).

The present invention also provides a method for screening C-terminaltruncated C5a peptides to identify immunogens for the production ofanti-C5a antibodies. In one embodiment, the method comprises, providinga C-terminal truncated C5a peptide, modifying the amino acid sequence ofsaid C-terninal truncated C5a peptide, and screening said C-terminaltruncated C5a peptide to identify immunogens for the production ofanti-C5a antibodies. In one embodiment, the C-terminal truncated C5apeptide which is provided is selected from SEQ ID NO:2, SEQ ID NO:4, SEQID NO:5, SEQ ID NO:14, SEQ ID NO: 15, and SEQ ID NO:16. In otherembodiments, the screening step involves a chemotaxis assay (See e.g.Examples 7, 8 and 11). In a different embodiment, the screening stepinvolves a competitive binding assay (See e.g. Examples 10 and 11). Inan additional embodiment, the screening step involves administering theC-terminal truncated peptides to septic animals (See e.g. Example 11).

DESCRIPTION OF THE FIGURES

FIG. 1 shows survival curves of septic rats with and without theadministration of anti-C5a antibodies.

FIG. 2 shows a graph demonstrating the ability of anti-C5a antibodies toreduce bacteria blood of septic rats.

FIG. 3 shows a graph demonstrating the ability of anti-C5a antibodies toreduce bacteria in the organs of septic rats.

FIG. 4 shows a graph demonstrating the ability of anti-C5a antibodies toincrease the level of H₂O₂ production in neutrophils of septic rats.

FIG. 5 shows a graph demonstrating the ability of synthetic peptides toreduce human C5a-induced chemotaxis of neutrophils.

FIG. 6 shows the chemotactic activity of KLH-linked synthetic peptidesof human C5a peptide.

FIG. 7 shows polyclonal rabbit anti-human C5a reactivity with regions ofhuman C5a peptide.

FIG. 8 shows the amino acid sequence of human C5a peptide and varioussmaller portions of the human C5a peptide.

FIG. 9 shows a graph demonstrating the ability of certain syntheticpeptides to inhibit the binding of human C5a peptide to humanneutrophils.

FIG. 10 shows inhibition of the H₂O₂ response to PMA by C5a peptide.

FIG. 11 shows a Western Blot demonstrating specificity of anti-human C5aantibodies for human C5a peptide.

DEFINITIONS

The phrase “symptoms of sepsis” refers to any symptoms characteristic ofa subject with sepsis including but not limited to, arterialhypotension, metabolic acidosis, fever, decreased systemic vascularresistance, tachypnea and organ dysfunction. Sepsis can result fromsepticemia (i.e., organisms, their metabolic end-products or toxins inthe blood stream), including bacteremia (i.e., bacteria in the blood),as well as toxemia (i.e., toxins in the blood), including endotoxemia(i.e., endotoxin in the blood). The term “sepsis” also encompassesfungemia (i.e., fungi in the blood), viremia (i.e., viruses or virusparticles in the blood), and parasitemia (i.e., helminthic or protozoanparasites in the blood). Thus, phenotypes associated with septicemia andseptic shock (acute circulatory failure resulting from septicemia oftenassociated with multiple organ failure and a high mortality rate) aresymptoms of sepsis.

The phrase “reduces the symptoms of sepsis” refers to a qualitative orquantitative reduction in detectable symptoms, including but not limitedto a detectable impact on the rate of recovery from disease.

The phrase “at risk for sepsis” in reference to a subject is hereindefined as a subject predisposed to the development of sepsis by virtueof the subject's medical status, including but not limited to suchfactors as infection, trauma (e.g., abdominal perforation, such as by agun shot wound), surgery (e.g., intestinal surgery), and invasiveprocedures (e.g., placement of a catheter, etc.) and the like.

As used herein, the term “antigen” refers to any agent (e.g., anysubstance, compound, molecule [including macromolecules], or othermoiety), that is recognized by an antibody, while the term “immunogen”refers to any agent (e.g., any substance, compound, molecule [includingmacromolecules], or other moiety) that can elicit an immunologicalresponse in an individual. These terms may be used to refer to anindividual macromolecule or to a homogeneous or heterogeneous populationof antigenic macromolecules. It is intended that the term encompassesprotein and peptide molecules or at least one portion of a protein orpeptide molecule, which contains one or more epitopes. In many cases,antigens are also immunogens, thus the term “antigen” is often usedinterchangeably with the term “immunogen.” The substance may then beused as an antigen in an assay to detect the presence of appropriateantibodies in the serum of the immunized animal.

The term “specific for” when used in reference to the interaction of anantibody and a protein or peptide means that the interaction isdependent upon the presence of a particular structure (i.e., theantigenic determinant or epitope) on the protein; in other words theantibody is recognizing and binding to a specific protein structurerather than to proteins in general (i.e. non-specific or backgroundbinding).

The term “not reactive with” when used in reference to the potentialinteraction of an antibody and a protein or peptide means that theantibody does not recognize or bind specifically to that particularprotein (i.e. binding is at background levels).

The term “operably linked” refers to an arrangement of elements whereinthe components so described are configured so as to perform their usualfunction. Thus, control sequences operably linked to a coding sequenceare capable of effecting the expression of the coding sequence. Thecontrol sequences need not be contiguous with the coding sequence, solong as they function to direct the expression As used herein, thephrase “anti-C5a antibody” refers to antibodies which are specific forcomplement component C5a peptide, or portions thereof.

As used herein, the term “adjuvant” is defined as a substance known toincrease the immune response to other antigens when administered withother antigens. If adjuvant is used, it is not intended that the presentinvention be limited to any particular type of adjuvant—or that the sameadjuvant, once used, be used all the time. It is contemplated thatadjuvants may be used either separately or in combination. The presentinvention contemplates all types of adjuvant, including but not limitedto agar beads, aluminum hydroxide or phosphate (alum), IncompleteFreund's Adjuvant, as well as Quil A adjuvant commercially availablefrom Accurate Chemical and Scientific Corporation, Gerbu adjuvant alsocommercially available (GmDP; C.C. Biotech Corp.), and bacterin (i.e.,killed preparations of bacterial cells).

As used herein, the term “N-terminal region of C5a peptide” refers tothe N-terminal 50% of the complement component C5a peptide.

As used herein, the term “C-terminal region of C5a peptide” refers tothe C-terminal 30% of the complement component C5a peptide.

As used herein, the term “wherein said antibody is not reactive with theC-terminal of C5a region” refers to antibodies that do not recognize orbind to the C-terminal 30% of the C5a peptide.

As used herein, the term “C-terminal truncated C5a peptides” refers topeptides of varying lengths derived from the N-terminal 70% of the C5apeptide, which do not include amino acid sequences from the C-terminal30% of the C5a peptide. Examples of these peptides include, but are notlimited to, SEQ ID NO:2 (from Rat C5a peptide), and SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16 (from Human C5apeptide).

As used herein, the terms “C5a peptide”, “C5a protein”, and “complementcomponent C5a peptide” all refer to the complement component peptide inanimals which is cleaved from the amino terminus of complement componentC5 when the complement system is activated. Examples of animals withthis protein include, but are not limited to, mice, rats, cows, pigs,and humans. This definition also includes peptides with syntheticsequences which share substantial homology to naturally occurring C5apeptides. An example of this type of sequence, includes, but is notlimited to, the sequence disclosed in Mandecki W, et al., Proc Natl AcadSci USA. June; 82(1l):3543-7(1985).

As used herein, the term “modifying the amino acid sequence” of saidC-terminal truncated C5a peptide refers to the addition, deletion, orsubstitution of one or more amino acids to create a variant or modifiedC-terminal truncated C5a peptide (See section II.b, below). Examples ofsuch variants or modified sequences are listed in Table 3 below.

A “variant” of a C5a peptide (or C-terminal truncated C5apeptide) isdefined as an amino acid sequence which differs by one or more aminoacids from the C5a peptide (or C-terminal truncated C5a peptide)sequence. The variant may have “conservative” changes, wherein asubstituted amino acid has similar structural or chemical properties,e.g replacement of leucine with isoleucine. More rarely, a variant mayhave “nonconservative” changes, e.g. replacement of glycine with atryptophan. Similar minor variations may also include amino aciddeletions or insertions (i.e., additions), or both. A variant may be anepitope as short as four amino acids in length, and as long as amodified full-lenth C5a peptide. More preferably, a variant is greaterthan five amino acids in length, and less than twenty-five amino acidsin length. Variants may also contain a fusion protein. In such cases,the variant may have more amino acids than the natural, full-lenght C5apeptide.

DESCRIPTION OF THE INVENTION

The present invention relates to compositions and methods for theprevention treatment of blood-borne and toxin mediated diseases, and inparticular anti-C5a antibodies for the prevention and treatment ofsepsis caused by various types of organisms in humans as well as otheranimals. It is contemplated that the present invention finds use in thetreatment of gram-negative and gram-positive sepsis. Although theinvention may be used for treatment of sepsis due to an individualorganism, it may also be used to treat sepsis caused by multipleorganisms (e.g., sepsis and/or bacteremia due to gram-negative andgram-positive organisms).

I. The C5a Peptide and Sepsis

The complement system is a complex group of proteins present in bodyfluids that, working together with antibodies or other factors, plays animportant role as mediators of immune, allergic, immunochemical andimmunopathological reactions. Activation of the complement system canresult in a wide range of reactions such as lysis of various kinds ofcells, bacteria and protozoa, inactivation of viruses, and the directmediation of inflammatory processes. Through the hormone-like activityof several of its components, the complement system can recruit andenlist the participation of other humoral and cellular effector systems.These in turn can induce directed migration of leukocytes, triggerhistamine release from mast cells, and stimulate the release oflysosomal constituents from phagocytes.

The complement system consists of at least twenty distinct plasmaproteins capable of interacting with each other, with antibodies, andwith cell membranes. Many of these proteins, when activated, combinewith still others to form enzymes that cleave and activate still otherproteins in the system. The sequential activation of these proteinsfollows two main pathways, the classical pathway and the alternativepathway. Both pathways use a common terminal trunk that leads to celllysis or virus inactivation.

The classical pathway can be activated by antigen-antibody complexes,aggregated immunoglobulins and non-immunological substances such as DNAand trypsin-like enzymes. The classical pathway includes activation ofC1, C4, C2 and C3. These components can be grouped into two functionalunits: C1 or recognition unit; and C4, C2 and C3 or activation unit.Five additional components denominated C5, C6, C7, C8, and C9 define themembrane attack unit forming the terminal trunk common to both pathways.

C5a peptide, also called anaphylatoxin, is a complement componentpeptide which is cleaved from the amino terminus of component C5 whenthe complement system is activated. C5a peptide has been shown tostimulate contraction of smooth muscle, enhance vascular permeability,promote the synthesis and release of other mediators includingleukotrienes, prostaglandins, platelet-activating factor, and histamine.In vivo, C5a peptide results in the accumulation of polymorphonuclearleukocytes (PMN) (i.e. neutrophils) and marcrophages at the site ofinflammation, one of the hallmark events of an acute inflammatoryresponse. In vitro, C5a peptide is a potent chemotaxin for leukocytes,most notably PMN and macrophages, and it activates PMN causing them torelease a variety of hydrolytic enzymes and to generate oxygen radicals.These latter-phenomena are thought to be responsible not only for thekilling of microorganisms but for much of the tissue destruction thattakes place in inflammatory situations.

There is abundant evidence that in sepsis, complement activation,production of cytokines, and unregulated inflammatory responses occurs.It is well established in humans with sepsis that complement activationand complement consumption have occurred, as defined by loss of wholehemolytic activity of serum complement (CH50) and the presence of C5apeptide in serum [Koehl, J., Bitter-Suermann, D., Anaphylatoxins.Complement in health and disease., Edited by Whaley, K., Loos, M.,Weiler, J. M., Kluwer Academic publishers, pp 299-324, (1993), andSolomkin, et al., Surgery 90:319-327, (1981)].

It is well established from in vitro studies that interaction of C5apeptide with C5a receptor (C5aR) leads to phosphorylation of serineresidues of the receptor, followed by rapid internalization of thereceptor-ligand complex, dephosphorylation of the receptor and itsrecycling back to the surface of the cell. All of this occurs fairlyrapidly. Furthermore, the maximal C5a-induced H₂O₂ response of theneutrophil requires that only a fraction of C5aR be occupied with ligand[Van Epps, et al., J. Immunol. 150:246-252 (1993)]. Neutrophilsstimulated with C5a peptide become refractory (“deactivated”) to furtherstimulation with this peptide; following exposure to high doses of C5apeptide, global deactivation to chemotactic peptides occurs [Ward, P. A.& Becker, E. L., J. Exp. Med. 127:693-709 (1968)]. There is clinicalevidence that blood neutrophils from humans with early sepsis losefunctional responsiveness to C5a peptide and in the latter phases ofsepsis lose responsiveness to structurally different chemotaxins such asthe bacterial chemotactic factor [Solomkin, J. S., et al., Surgery90:319-327 (1981)]. It has also been reported that C5 deficient micedemonstrate somewhat prolonged survival times when sepsis is induced,but ultimately all animals succumbed to the sepsis syndrome [Olson, L.M., et al., Ann. Surg. 202:771-776 (1985)].

It is not necessary to the successful practice the present inventionthat one understand the precise mechanism by which a therapeutic benefitis achieved, nor is the present invention limited to any particularmechanistic explanation. However, it is believed that sepsis results inexcessive production of C5a peptides, which leads to deactivation ofneutrophils, compromising the respiratory burst (H₂O₂ production) ofthese cells and the closely linked bactericidal function, which isdependent upon H₂O₂ generation and participation of myeloperoxidase. Theanti-C5a antibodies of the present invention, therefore, are believed toprevent the deactivation of neutrophils caused by sepsis, thuspreserving the bactercidial function of the neutrophils. In this regard,the present invention contemplates antibodies specific for complementcomponent C5a peptides and methods of using these antibodies to treatsepsis. In some embodiments, these antibodies are specific forcomplement component C5a peptide, wherein said C5a peptide has aC-terminal region and an N-terminal region, and wherein said antibody isnot reactive with said C-terminal region.

II. Generating Antibodies to C5a Peptides

a. Antibodies

The present invention contemplates monoclonal, polyclonal, and humanizedantibodies to C5a peptides. In some embodiments, the antibodies arespecific for complement component C5a peptide, wherein said C5a peptidehas a C-terminal region and an N-terminal region, and said antibody isnot reactive with said C-terminal region.

Monoclonal antibodies useful in this invention are obtained, forexample, by well known hybridoma methods. In one embodiment, an animalis immunized with a preparation containing C-terminal truncatedpeptides. A fused cell hybrid is then formed between antibody-producingcells from the immunized animal and an immortalizing cell such as amyeloma. In one embodiment, antibodies of the present invention areproduced by murine hybridomas formed by fusion of mouse myeloma orhybridoma which does not secrete antibody with murine spleen cells whichsecrete antibodies obtained from mice immunized against C-terminaltruncated C5a peptides.

In some embodiments, mice are immunized with a primary injection ofC-terminal truncated C5a peptides, followed by a number of boostinginjections. During or after the immunization procedure, sera of the micemay be screened to identify mice in which a substantial immune responseto the C-terminal truncated C5a peptides has been evoked. From theselected mice, spleen cells are obtained and fusions are preformed.Suitable fusion techniques include, but are not limited to, the Sendaivirus technique [Kohler, G. and Milstein, C., Nature 256:495 (1975)] orthe polyethylene glycol method [Kennet, R. H., “Monoclonal Antibodies,Hybridoma—A New Dimension in Biological Analysis,” Plenum Press, NY(1980)].

The hybridomas are then screened for production of anti-C5a antibodies.Suitable screening techniques include, but are not limited to, solidphase radioimmunoassay. A solid phase immunoadsorbent is prepared bycoupling C5a peptides to an insoluble matrix. The immunoadsorbent isbrought into contact with culture supernatants of hybridomas. After aperiod of incubation, the solid phase is separated from thesupernatants, then contacted with a labelled antibody against murineimmunoglobulin. Label associated with the immunoadsorbent indicates thepresence of hybridoma products reactive with C5a peptides.

In preferred embodiments the monoclonal anti-C5a antibodies are producedin large quantities by injecting anti-C5a antibody producing hybridomacells into the peritoneal cavity of mice and, after an appropriate time,harvesting acites fluid from the mice which yield a high titer ofhomogenous antibody. The monoclonal antibodies are isolated therefrom.Alternatively, the antibodies are produced by culturing anti-C5aantibody producing cells in vitro and isolating secreted monoclonalanti-C5a antibodies from the cell culture medium directly.

Another method of forming antibody-producing cells is by viral oroncogenic transformation. For example, a B-lymphocyte which producesanti-C5a specific antibody is infected and transformed with a virus,such as the Epstein-Barr virus, to give an immortal antibody-producingcell [Kozbon and Roder, Immunol. Today 4:72-279 (1983)].

The present invention also contemplates anti-C5a polyclonal antibodies.Polyclonal antibodies can be prepared by immunizing an animal with acrude preparation of C-terminal truncated C5a peptides, or purifiedC-terminal truncated C5a peptides. The animal is maintained underconditions whereby antibodies reactive with the components of thepeptides are produced. [See e.g. Elzaim, et al., Infect. Immun. May;66(5):2170-9 (1998)]. Typically the animal is “boosted” by additionalimmunizations to increase the antibody titer. In one method, blood iscollected from the animal upon reaching a desired titer of antibodies.The serum containing the polyclonal antibodies (antisera) is separatedfrom the other blood components. The polyclonal antibody-containingserum may be further separated into fractions of particular types ofantibodies (e.g. IgG or IgM) or monospecific antibodies can be affinitypurified from polyclonal antibody containing serum. In another method,the immunized animal is a bird. In this method antibodies (IgY) arecollected from egg yolks. The egg yolk is separated from the yolk lipidand non-antibody proteinaceous matter, recovering the IgY anti-C5aantibodies in purified form (see e.g. U.S. Pat. No. 4,357,272 to Polsonand U.S. Pat. No. 5,904,922 to Carroll).

The present invention also contemplates humanized antibodies (i.e.substantially non-immunogenic antibodies). Such antibodies areparticularly useful in treating human subjects. Chimeric and ‘reshaped’humanized anti-C5a antibodies may be produced according to techniquesknown in the art (see e.g. U.S. Pat. No. 5,585,089 to Queen et al., andKettleborough, et al., Protein Engineering, vol. 4, no.7, pp 773-783,1991). In one embodiment, humanized anti-C5a chimeric antiboides areproduced using a combinatorial approach (see e.g. U.S. Pat. No.5,565,332 to Hoogenboom et al. and U.S. Pat. No. 5,658,727 to Barbas etal.). The present invention also contemplates single polypeptide chainbinding molecules which have binding specificity and affinitysubtantially similar to the binding specificity and affinity of thelight and heavy chain aggregate variable region of an anti-C5a antibody(see e.g. U.S. Pat. No. 5,260,203 to Ladner et al.).

b. C5a Peptide Immunogens

The present invention provides various C5a peptide immunogens. Forexample, the C5a peptides can be from various animals (e.g. human, rat,pig, and cow). The amino acid sequence of these C5a peptides aredescribed in the literature [see Rothermel et al., Biochim. Biophys.Acta 1351 (1-2), 9-12, (1997) {rat}; Babkina, I. N., et al., BioorgKhim, May;21(5):359-64, (1995){human}; Gerard, C. et al., J. Biol Chem.255(10), 4710-4715, (1980){pig}; and Zarbock, J., et al., FEBS Lett.238(2), 289-294, (1988){cow}]. The C5a immunogen may be the full lengthC5a peptide, or various peptides derived from the full length C5apeptide. In particular embodiments, the peptides are C-terminaltruncated peptides (e.g. SEQ ID NOS:2, 4, 5, 14, 15 and 16).Representative sequences are listed in Table 1. Representative human andrat DNA sequences which are used to generate various C-terminaltruncated C5a peptides are listed in Table 2, along with the full humanand full rat C5a DNA sequences. Modifications of these sequences (i.e.longer/shorter sequence, from various regions) are contemplated by thepresent invention. Generation of these various C-terminal truncated C5apeptide immunogens are described below.

Variants of the C-terminal truncated C5a peptides are contemplated asuseful immunogens (See e.g. Table 3). For example, it is contemplatedthat an isolated replacement of a leucine with an isoleucine or valine,an aspartate with a glutamate, a threonine with a serine, or a similarreplacement of an amino acid with a structurally related amino acid(i.e., conservative mutations) will not have a major effect on thebiological activity of the resulting molecule. Conservative replacementsare those that take place within a family of amino acids that arerelated in their side chains. Genetically encoded amino acids can bedivided into four families: (1) acidic (aspartate, glutamate); (2) basic(lysine, arginine, histidine); (3) nonpolar (alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan); and (4)uncharged polar (glycine, asparagine, glutamine, cysteine, serine,threonine, tyrosine). Phenylalanine, tryptophan, and tyrosine aresometimes classified jointly as aromatic amino acids. In similarfashion, the amino acid repertoire can be grouped as (1) acidic(aspartate, glutamate); (2) basic (lysine, arginine histidine), (3)aliphatic (glycine, alanine, valine, leucine, isoleucine, serine,threonine), with serine and threonine optionally be grouped separatelyas aliphatic-hydroxyl; (4) aromatic (phenylalanine, tyrosine,tryptophan); (5) amide (asparagine, glutamine); and (6) sulfur-containing (cysteine and methionine) (See e.g., Stryer ed.,Biochemistry, 2nd ed,, WH Freeman and Co.[1981]).

Thus, in certain embodiments, modifications of the C-terminal truncatedC5a peptides are contemplated by the present invention. Similar minorvariations may also include amino acid deletions or insertions (i.e.additions), or both. Guidance in determining which and how many aminoacid residues may be substituted, inserted or deleted without abolishingbiological or immunological activity may be found using computerprograms well known in the art, for example, DNAStar software or GCG(Univ. of Wisconsin).

Whether a change in the amino acid sequence of a C-terminal truncatedC5a peptide results in a useful immunogen for producing the anti-C5aantibodies of the present invention can be readily determined. Onemethod involves screening the C-terminal truncated C5a peptides for theability to inhibit the chemotaxis of neutrophils. Useful immunogens areidentified by the ability to induce chemotaxis (See e.g. Examples 7, 8and 11). Another indication of a useful immunogen is the ability of theC-terminal truncated C5a peptide to inhibit chemotaxis when combinedwith C5a peptide (See e.g. Examples 7, 8, and 11). Another methodinvolves screening the C-terminal truncated C5a peptides for the abilityto antagonize the binding of labelled C5a peptides to neutrophils in acompetitive assay (See e.g. Examples 10 and 11). Yet another methodinvolves administering the C-terminal truncated C5a peptides to CLP issepsis induced rats, and monitoring their response over a given timeperiod. Useful immunogens are identified by the ability to reduce thesymptoms of sepsis, and/or increase survival times of the rats (See e.g.Example 11).

The C-terminal truncated C5a peptides employed in the present inventionmay also comprise a fusion partner. Examples of fusion partners includeProtein A, ABP, GST, poly histidine, HA, KLH, and MBP. Other fusionpartners are well known in the art. (See Nilsson et al., Prot. Expr.Purif., 11(1):1-16 [1997]). The fusion partner may serve variousfunctions, including, but not limited to, enhancement of the solubilityof the C-terminal truncated C5a peptides, as well as providing an“affinity tag” to allow the purification of the recombinant fusionC-terminal truncated C5a peptide from the host cell or culturesupematant, or both. If desired, the exogenous protein fragment may beremoved from the peptide of interest prior to immunization by a varietyof enzymatic or chemical means known in the art.

In some embodiments, nucleic acid sequences corresponding to thesevarious C-terminal truncated C5a peptides (e.g., SEQ ID NOS:10, 11, 13,17, 18, and 19) are used to generate recombinant DNA molecules thatdirect the expression of the C-terminal truncated C5a peptides inappropriate host cells, which are then purified and used as immunogensto generate the antibodies of the present invention. These DNA sequencesmay be included in any one of a variety of expression vectors forexpressing C-terminal truncated C5a peptides in various hosts. Examplesof vectors include, but are not limited to, chromosomal, nonchromosomaland synthetic DNA sequences, e.g., derivatives of SV40; bacterialplasmids; phage DNA; baculovirus; yeast plasmids; vectors derived fromcombinations of plasmids and phage DNA, viral DNA such as vaccinia,adenovirus, fowl pox virus, and pseudorabies, and the like. Any vectormay be used as long as it is replicable and viable in the host.

Large numbers of suitable vectors are known to those of skill in theart, and are commercially available. Such vectors include, but are notlimited to, the following: 1) Bacterial—pQE70, pQE60, pQE-9 (Qiagen),pBS, pD10, phagescript, psiX174, pbluescript SK, pBSKS, pNH8A, pNH16a,pNH18A, pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5(Pharmacia); and 2) Eukaryotic—pWLNEO, pSV2CAT, pOG44, PXT1, pSG(Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia). In general, mammalianexpression vectors comprise an origin of replication, a suitablepromoter and enhancer, and also any necessary ribosome binding sites,polyadenylation site, splice donor and acceptor sites, transcriptionaltermination sequences, and 5′ flanking nontranscribed sequences. DNAsequences derived from the SV40 splice, and polyadenylation sites may beused to provide the required nontranscribed genetic elements.

The DNA sequence in the expression vector may be operably linked to anappropriate expression control sequence(s) (promoter) to direct mRNAsynthesis. Promoters useful in the present invention include, but arenot limited to, the LTR or SV40 promoter, the E. coli. lac or trp, thephage lambda PL and PR, T3 and T7 promoters, and the CMV immediateearly, HSV thymidine kinase, and mouse metallothionein-I promoters andother promoters known to control expression of peptides in prokaryoticor eukaryotic cells or their viruses. Recombinant expression vectorsgenerally include origins of replication and selectable markerspermitting transformation of the host cell (e.g., dihydrofolatereductase or neomycin resistance for eukaryotic cell culture, or such astetracycline or ampicillin resistance in E. coli).

Transcription of the DNA encoding the C-terminal truncated C5a peptidesof the present invention is increased by inserting an enhancer sequenceinto the vector. Enhancers are cis-acting elements of DNA, usually aboutfrom 10 to 300 bp that act on a promoter to increase its transcription.Enhancers useful in the present invention include, but are not limitedto, the SV40 enhancer on the late side of the replication origin bp 100to 270, a cytomegalovirus early promoter enhancer, the polyoma enhanceron the late side of the replication origin, and adenovirus enhancers.The expression vector may also contain a ribosome binding site fortranslation initiation and a transcription terminator.

Various host cells may be employed to recombinantly express theC-terminal truncated C5a peptides. Suitable host cells are highereukaryotic cells (e.g., a mammalian or insect cell), lower eukaryoticcells (e.g., a yeast cell), and prokaryotic cells (e.g., a bacterialcell). Specific examples of host cells include, but are not limited to,Escherichia coli, Salmonella typhimurium, Bacillus subtilis, and variousspecies within the genera Pseudomonas, Streptomyces, and Staphylococcus,as well as, Saccharomycees cerivisiae, Schizosaccharomycees pombe,Drosophila S2 cells, Spodoptera Sf9 cells, Chinese Hamster Ovary (CHO)cells, COS-7 lines of monkey kidney fibroblasts, (Gluzman, Cell, 23:175[1981]), C127, 3T3, HeLa and BHK cell lines.

The constructs in host cells can be used in a conventional manner toproduce the C-terminal truncated C5a peptides encoded by the recombinantsequences. In some embodiments, introduction of the construct into thehost cell is effected by calcium phosphate transfection, DEAE-Dextranmediated transfection, or electroporation [Davis et al., Basic Methodsin Molecular Biology, (1986)].

Following transformation of a suitable host strain and growth of thehost strain to an appropriate cell density, the selected promoter isinduced by appropriate means (e.g., temperature shift or chemicalinduction) and cells are cultured for an additional period. Cells aretypically harvested by centrifugation, disrupted by physical or chemicalmeans, and the resulting crude extract retained for furtherpurification. Methods for recovering and purifying C-terminal truncatedC5a peptides from recombinant cell culture include, but are not limitedto, ammonium sulfate or ethanol precipitation, acid extraction, anion orcation exchange chromatography, phosphocellulose chromatography,hydrophobic interaction chromatography, affinity chromatography,hydroxylapatite chromatography and lectin chromatography. Highperformance liquid chromatography (HPLC) can also be employed for finalpurification steps.

DNA sequences having coding sequences (e.g., SEQ ID NOS:10, 11, 13, 17,18, and 19) fused in frame to a marker sequence allow for purificationof the C-terminal truncated C5a peptide. One example is a histidine tagwhich may be supplied by a vector (e.g., a pQE-9 vector) which providesfor purification of the polypeptide fused to the marker in the case of abacterial host, or, for example, the marker sequence may be ahemagglutinin (HA) tag when a mammalian host (e.g., COS-7 cells) isused. The HA tag corresponds to an epitope derived from the influenzahemagglutinin protein (Wilson, et al., Cell, 37:767 [1984]).

The coding sequences for the C-terminal truncated C5a peptides can alsobe incorporated as a part of a fusion gene including a nucleotidesequence encoding a different polypeptide. One example employs the VP6capsid protein of rotavirus used as an immunologic carrier protein forthe C-terminal truncated C5a peptides, either in the monomeric form orin the form of a viral particle. The nucleic acid sequencescorresponding to the various C5a peptides to which antibodies are to beraised can be incorporated into a fusion gene construct which includescoding sequences for a late vaccinia virus structural protein to producea set of recombinant viruses expressing fusion proteins comprisingC-terminal truncated C5a peptides as part of the virion. It has beendemonstrated with the use of immunogenic fusion proteins utilizing theHepatitis B surface antigen fusion proteins that recombinant Hepatitis Bvirions can be utilized in this role as well. Similarly chimericconstructs coding for fusion proteins containing C-terminal truncatedC5a peptides and the poliovirus capsid protein are created to enhanceimmunogenicity of the set of polypeptide antigens [see e.g., EPPublication No. 025949; and Evans et al., Nature 339:385 (1989); Huanget al., J. Virol. 62:3855 (1988); and Schlienger et al., J. Virol. 66:2(1992)]. The Multiple Antigen Peptide system for peptide-basedimmunization can also be utilized, wherein a desired C-terminaltruncated C5a peptide sequence is obtained directly from organo-chemicalsynthesis of the peptide onto an oligomeric branching lysine core [seee.g., Posnett et al., JBC 263:1719 (1988) and Nardélli et al., J.Immunol. 148:914 (1992)]. C-terminal truncated C5a peptides can also beexpressed and presented by bacterial cells in order to generate theantibodies of the present invention.

In addition to utilizing fusion proteins to enhance immunogenicity,fusion proteins can also facilitate the purification of proteins.Accordingly, the C5a peptides can be generated as aglutathione-S-transferase (GST) fusion protein. Such GST fusion proteinsenable easy purification of the C5a peptides, such as by the use ofglutathione-derivatized matrices [see e.g, Current Protocols inMolecular Biology, Eds. Ausabel et al., N.Y.: John Wiley & Sons,(1991)]. Also, a fusion gene coding for a purification leader sequence,such as a poly-(His)/enterokinase cleavage site sequence at theN-terminus of the desired C-terminal truncated C5a peptide, can allowpurification of the expressed C-terminal truncated C5a fusion protein byaffinity chromatography using, for example, a Ni2+ metal resin. Thepurification leader sequence can then be subsequently removed bytreatment with enterokinase [see e.g., Hochuli et al., J. Chromatography411:177 (1987)].

Techniques for making fusion genes are well known. Essentially, thejoining of various DNA fragments coding for different polypeptidesequences is performed in accordance with conventional molecular biologytechniques, employing blunt-ended or stagger-ended termini for ligation,restriction enzyme digestion to provide for appropriate termini,filling-in of cohesive ends as appropriate, alkaline phosphatasetreatment to avoid undesirable joining, and enzymatic ligation. Thefusion gene is synthesized by conventional techniques includingautomated DNA synthesizers. Alternatively, in other embodiments of thepresent invention, PCR amplification of gene fragments can be carriedout using anchor primers which give rise to complementary overhangsbetween two consecutive gene fragments which is subsequently annealed togenerate a chimeric gene sequence [see e.g., Current Protocols inMolecular Biology, Eds. Ausubel et al., John Wiley & Sons (1992)].

The C-terminal truncated C5a peptide sequences may be synthesized, wholeor in part, using chemical methods well known in the art [see e.g.,Caruthers et al., Nuc. Acids Res. Symp. Ser. 7:215-233 (1980); Crea andHorn, Nuc. Acids Res. 9:2331 (1980); Matteucci and Caruthers,Tetrahedron Lett 21:719 (1980); and Chow and Kempe, Nuc. Acids Res.9:2807-2817 (1981)]. For example, C-terminal truncated C5a peptides canbe synthesized by solid phase techniques, cleaved from the resin, andpurified by preparative high performance liquid chromatography [seee.g., Creighton (1983) Proteins Structures And Molecular Principles, W HFreeman and Co, New York N.Y.]. In other embodiments of the presentinvention, the composition of the synthetic peptides may be confirmed byamino acid analysis or sequencing (see e.g., Creighton, supra).

Direct peptide synthesis can be performed using various solid-phasetechniques [Roberge et al., Science 269:202-204 (1995)] and automatedsynthesis may be achieved, for example, using ABI 431A PeptideSynthesizer (Perkin Elmer, Norwalk Conn.) in accordance with theinstructions provided by the manufacturer. Additionally the amino acidsequence of the C-terminal truncated C5a peptide sequences may bealtered during direct synthesis yielding various C-terminal truncatedC5a peptides which are used to generate the anti-C5a antibodies of thepresent invention.

Compounds mimicking the necessary conformation for generating theantibodies of the present invention are also contemplated as within thescope of the invention. For example, mimetics of the C-terminaltruncated C5a peptides are contemplated. A variety of designs for suchmimetics are possible. For example, cyclic C-terminal truncated C5apeptides, in which the necessary conformation for immunogenicity isstabilized by non-peptides, are specifically contemplated (See e.g. U.S.Pat. No. 5,192,746 to Lobl, et al., U.S. Pat. No. 5,169,862 to Burke,Jr., et al., U.S. Pat. No. 5,539,085 to Bischoff, et al., U.S. Pat. No.5,576,423 to Aversa et al., U.S. Pat. No. 5,051,448 to Shashoua, andU.S. Pat. No. 5,559,103 to Gaeta et al., all of which describe muliplemethods for creating such compounds). The present invention alsocontemplates non peptide compounds that mimic C-terminal truncated C5apeptides, as well as multimeric compounds that repeat the relevantpeptide sequences.

Table 1 C5a Sequences and Peptides Useful in Generating AntibodiesSpecies SEQ ID NO: Amino Acid Sequence Rat C5a (full seq.) SEQ ID NO:1DLQLLHQKVEEQAAKYKHRVP KKCCYDGARENKYETCEQRVA RVTIGPHCIRAFNECCTIADKIRKESHHKGMLLGR Rat C5a (residues 17-36) SEQ ID NO:2 KHRVPKKCCYDGARENKYETHuman C5a (full seq.) SEQ ID NO:3 MLQKKIEEIAAKYKHSVVKKCCYDGASVNNDETCEQRAARISLG PRCIKAFTECCVVASQLRANISH KDMQLGR Human C5a (res.1-20) SEQ ID NO:4 MLQKKIEEIAAKYKHSVVKK Human C5a (res. 21-40) SEQ IDNO:5 CCYDGASVNNDETCEQRAAR Human C5a (res. 55-74) SEQ ID NO:6CVVASQLRANISHKDMQLGR Human C5a (res. 12-20) SEQ ID NO:14 KYKHSVVKK HumanC5a (res. 28-33) SEQ ID NO:15 VNNDET Human C5a (res. 38-46) SEQ ID NO:16AARISLGPR Bovine C5a (full seq.) SEQ ID NO:7 MLKKKIEEEAAKYRNAWVKKCCYDGAHRNDDETCEERAARIAI GPECIKAFKSCCAIASQFRADEH HKNMQLGR Porcine C5a(full seq.) SEQ ID NO:8 MLQKKIEEEAAKYKYAMLKKC CYDGAYRNDDETCEERAARIKIGPKCVKAFKDCCYIANQVRAEQ SHKNIQLGR

TABLE 2 Rat and Human C5a Polynucleotide Sequences Useful in GeneratingC5a Peptides Species SEQ ID NO: Polynucleotide Seciuence Human C5a (fullseq.) SEQ ID NO:9 GATCCAGTATGTTGCAAAAAA AAATTGAAGAAATTGCTGCTAAATATAAACATTCTGTTGTTAA AAAATGTTGTTATGATGGAGCT TCTGTTAATAATGATGAAACCTGCGAACAACGCGCTGCTAGAA TTTCTTTGGGACCTAGATGTA TTAAAGCATTTACAGAATGTTGTGTTGTTGCTTCTCAATTGAG GCGAATATTTCTCATAAAGATA TGCAATTGGGAAGATAGGATCCGTCGA Human C5a (for res. 1-20) SEQ ID NO:10 ATGTTGCAAAAAAAAATTGAAGAAATTGCTGCTAAATA TAAACATTCTGTTGTTAAAAAA Human C5a (for res. 21-40)SEQ ID NO:11 TGTTGTTATGATGGAGCTTC TGTTAATAATGATGAAACCTGCGAACAACGCGCTGCTAGA Human C5a (for res. 12-20) SEQ ID NO:17TTGCTGCTAAATATAAACAT TCTGTTG Human C5a (for res. 28-33) SEQ ID NO:18GAGCTTCTGTTAATAATG Human C5a (for res. 38-46) SEQ ID NO:19AACAACGCGCTGCTAGAATT TCTTTGG Rat C5a (full seq.) SEQ ID NO:12GACCTGCAGCTCCTGCATCAG AAAGTGGAAGAACAAGCTGCT AAATACAAACACCGTGTGCCCAAGAAATGCTGTTATGATGGA GCCCGAGAAAACAAATACGAA ACCTGTGAGCAGCGAGTTGCCCGGGTGACCATAGGCCCACAC TGCATCAGGGCCTTCAACGAG TGTTGTACTATTGCGGATAAGATCCGAAAAGAAAGCCACCAC AAAGGCATGCTGTTGGGAAGG Rat C5a (for res. 17-36) SEQID NO:13 AAACACCGTGTGCCCAAGAAA TGCTGTTATGATGGAGCCCGA GAAAACAAATACGAAACC

TABLE 3 Variant C-Terminal Truncated C5a Peptide Sequences SEQ ID NO:Amino Acid Sequence SEQ ID NO:20 KYKHTVVKK SEQ ID NO:21 KYKHSAVKK SEQ IDNO:22 KYKHSAAKK SEQ ID NO:23 KYKHSVAKK SEQ ID NO:24 VNNQET SEQ ID NO:25VNNDES SEQ ID NO:26 VNNQES SEQ ID NO:27 ANNDET SEQ ID NO:28 AARISIGPRSEQ ID NO:29 AARISVGPR SEQ ID NO:30 AARITLGPR SEQ ID NO:31 AVRISLGPR SEQID NO:32 VARISLGPR SEQ ID NO:33 VVRISLGPR SEQ ID NO:34MLQKKIEEIAAKYKHSVVK SEQ ID NO:35 MLQKKIEEIAAKYKHSVV SEQ ID NO:36MLQKKIEEIAAKYKHSV SEQ ID NO:37 MLQKKIEEIAAKYKHS SEQ ID NO:38MLQKKIEEIAAKYKH SEQ ID NO:39 LQKKIEEIAAKYKHSVVKK SEQ ID NO:40QKKIEEIAAKYKHSVVKK SEQ ID NO:41 KKIEEIAAKYKHSVVKK SEQ ID NO:42KIEEIAAKYKHSVVKK SEQ ID NO:43 IEEIAAKYKHSVVKK SEQ ID NO:44MIQKKIEEIAAKYKHSVVKK SEQ ID NO:45 MVQKKIEEIAAKYKHSVVKK SEQ ID NO:46MLDKKIEEIAAKYKHSVVKK SEQ ID NO:47 MLQKKIEEIAAKYKHTVVKK SEQ ID NO:48MLQKKIEEIVAKYKHSVVKK SEQ ID NO:49 MLQKKIEEIVVKYKHSVVKK SEQ ID NO:50MLQKKIEEIAAKYKHSVAKK SEQ ID NO:51 MLQKKIEEIAAKYKHSAAKK SEQ ID NO:52MLQKKIEEIAAKYKHSAVKK SEQ ID NO:53 MLQKKIEEIAVKYKHSVVKK SEQ ID NO:54CCYDGASVNNDETCEQRAA SEQ ID NO:55 CCYDGASVNNDETCEQRA SEQ ID NO:56CCYDGASVNNDETCEQR SEQ ID NO:57 CCYDGASVNNDETCEQ SEQ ID NO:58CCYDGASVNNDETCE SEQ ID NO:59 CYDGASVNNDETCEQRAAR SEQ ID NO:60YDGASVNNDETCEQRAAR SEQ ID NO:61 DGASVNNDETCEQRAAR SEQ ID NO:62GASVNNDETCEQRAAR SEQ ID NO:63 ASVNNDETCEQRAAR SEQ ID NO:64CCYQGASVNNDETCEQRAAR SEQ ID NO:65 CCYDGASVNNQETCEQRAAR SEQ ID NO:66CCYQGASVNNQETCEQRAAR SEQ ID NO:67 CCYDGASVNNDESCEQRAAR SEQ ID NO:68CCYDGATVNNDETCEQRAAR SEQ ID NO:69 CCYDGVSVNNDETCEQRAAR SEQ ID NO:70CCYDGASANNDETCEQRAAR SEQ ID NO:71 CCYDGASVNNDETCEQRVAR SEQ ID NO:72CCYDGASVNNDETCEQRVVR SEQ ID NO:73 CCYDGASVNNDETCEQRAVR SEQ ID NO:74CCYDGVSANNDETCEQRVVR

In one embodiment, the present invention contemplates a method ofproducing polyclonal antibodies, comprising; providing an animal and animmunogenic composition, wherein the composition comprises C-terminaltruncated C5a peptides; and immunizing the animal with the immunogeniccomposition in order to generate antibodies. It is not intended that thepresent invention be limited to particular C-terminal truncatedpeptides. A variety of C-terminal truncated peptides are contemplated.In one embodiment, the C-terminal truncated peptide corresponds to theentire N-terminal region of C5a peptide. In another embodiment, theC-terminal truncated peptide is a fragment or portion of the N-terminalregion of C5a peptide. In another embodiment, the fragment or portion ofthe N-terminal region of C5a peptide is between approximately 5 andapproximately 50 amino acids in length. In some embodiments, theC-terminal truncated peptide is fifty amino acids in length. In otherembodiments, the C-terminal truncated peptides are approximately fiveamino acids in length. In preferred embodiments, the C-terminaltruncated peptides are approximately 20 amino acids in length. Inespecially preferred embodiments, the C-terminal truncated peptides areselected from SEQ ID NOS:2, 4, and 5.

III. Antibody Applications

A. Prophylactic Use In Humans

The diagnosis of sepsis is problematic. First, the development of sepsisdoes not require the persistent release of toxin(s), nor the presence oforganisms, in the circulation. Thus, many patients who die of sepsis arenever shown to be bactermic. [R. C. Bone, Ann. Intern. Med. 115:457-469(1991)]. Second, even if bacteria are detected, the amount of timeneeded for this detection is often too great to be practical.

For these reasons and others, the present invention contemplates the useof anti-C5a antibodies in humans prior to the onset of symptoms (e.g.,prophylactically). In particular, the present invention contemplates theuse of anti-C5a antibodies as prophylactic treatment in patients at highrisk for infection, as well as sepsis.

High risk patients include surgical patients (particularly the elderly),low birth weight infants, bum victims and trauma patients. Traumapatients are particularly difficult to examine because of the multitudeof invasive procedures that they have undergone. Trauma patients arealso typically hooked up to a number of devices, including intravascularlines, mechanical ventilators and Foley catheters. While every attemptis made to change intravascular lines, this is frequently impossiblebecause of the extent of trauma and the lack of venous accessibility.[E. S. Caplan and N. Hoyt, Am. J. Med. 70:638-640 (1981)].

Most patients with multiple trauma have fever, as well as increasedwhite cell counts due to the stress of the trauma itself. The classicindicators of infection, therefore, may or may not reflect an ongoinginfection.

Because of this, current clinical practice involves treating patientswith antibiotics only for specific indications, and for as short aperiod of time as possible. Generally, the average course for anydocumented infection is seven to ten days. Prophylactic antibiotics areused in only three instances: open fractures, penetrating abdominalinjuries and penetrating facial injuries in which there is injury to therespiratory mucosa. Even in these situations, antibiotics are used foronly three to five days, depending on the injury.

Burn patients have many problems with respect to the diagnosis andtherapy for infection. Since the magnitude of thermal injury is relatedto the level of trauma in a burn victim, this becomes even more of aproblem with acute cases. It is reported that septicemia appears in theblood cultures of burn patients almost four days after a septic state.[M. Meek et al., J. Burn Care Rehab. 12:564-568 (1991)]. Consequently,therapy with the antibodies of the present invention is particularlyappropriate immediately after the bum injury as a means of preventing aseptic reaction. Furthermore, in severe cases, consideration should begiven to the topical administration of the antibodies of the presentinvention to prevent wound sepsis.

Finally, surgical patients also represent a risk group where theantibodies of the present invention can be used successfully. Currentpractice involves the prophylactic use of antibiotics in a very narrowcategory of cases (e.g., elective colorectal procedures,cholecystectomy, hysterectomy and Caesarean sections). [R. L. Nichols inDecision Making in Surgical Sepsis, B. C. Decker, Inc., Philadelphia,pp. 20-21 (1991)]. One to two grams of a broad-spectrum antibiotic areadministered intravenously at the induction of anesthesia. An additionaldose may be given during an extensive procedure or post-operatively butprophylaxis beyond 24 hours is not indicated. Twenty-four hours ofantibiotic prophylaxis is considered to be sufficient to controlcontamination. Continuance of antibiotic prophylaxis beyond 24 hours isan added expense, particularly when using an antibiotic with short serumand tissue half-lives. Most importantly, continuation of antibioticprophylaxis also runs an excessive risk of drug toxicity and emergenceof resistant strains. As such, the present invention contemplates theuse of anti-C5a antibodies to help reduce the need for antibiotics, andreduce the risk of sepsis.

In this regard, the present invention contemplates a method comprising;providing; a subject at risk for sepsis, and a therapeutic compositioncomprising an antibody specific for complement component C5a peptide,and prophylactically administering said therapeutic composition to thesubject. In some embodiments administering the composition prevents theonset of symptoms of sepsis.

B. Acute Therapy in Humans

The present invention also contemplates the use of anti-C5a antibodiesin a therapeutic preparation for acute treatment. In this case,treatment involves administration of the antibodies after infection isdetected and/or sepsis is suspected.

Evidence suggestive of infection includes the following: (1) coretemperature higher than 38° C. or lower than 35° C.; (2) peripheralblood leukocyte count greater than 12×10⁹/L or less than 3×10⁹/L (notdue to chemotherapy), or at least 20% immature forms; (3) growth ofgram-negative organisms from a blood culture drawn within the preceding48 hours; or (4) documented or suspected site of gram-negativeinfection.

A systemic septic reaction is characterized by at least one of thefollowing: arterial hypotension (systolic blood pressure <90 mm Hg or anacute drop of 30 mm Hg); metabolic acidosis (base deficit >5 mEq/L);decreased systemic vascular resistance (systemic vascular resistance<800 dynes/s cm⁵); tachypnea (respiratory rate >20/min orventilation >10 L/min if mechanically ventilated); or otherwiseunexplained dysfunction of the kidney (urine output <30 ml/h), or lungs.

It must be stressed that the anti-C5a antibodies of the presentinvention should ideally be used prior to a systemic infection, ifpossible. For example, the antibodies are administered immediately afterbacteremia or fungemia is detected. Similarly, antibodies can beadministered where there is an obvious sign of infection at a particularsite (e.g., wounds, sinusitis, meningitis, respiratory,gastrointestinal, or urinary tract infections, etc.).

Primary bacteremia is typically defined as two or more blood cultureswith the same bacterial organism occurring in a patient with no otherobvious site of infection. Sinusitis is diagnosed in a patient who hasat least two of the following: purulent nasal discharge,roentgenographic evidence of sinusitis or purulent material aspiratedfrom the sinuses.

The lower respiratory tract is a common site of infection. Pneumonia inthe intubated patient is diagnosed in a patient when there is fever,leukocytosis and a Gram stain with many polymorphonuclear leukocytes.Pneumonia may also be diagnosed in a patient with a new infiltrate thathas not cleared with intensive physical therapy (this last criterionhelps rule out atelectasis).

The C5a peptide has been implicated in the pathogenesis of bacterialmeningitis [Stahel, et al., J. Immunol. July 15;159(2):861-9 (1997)]. Assuch, treatment of acute meningitis with the anti-C5a antibodies of thepresent invention is contemplated. Among the bacterial causes ofmeningitis, two gram-negative organisms (Neisseria meningitidis andHaemophilus influenzae), and one gram-positive organism (Streptococcuspneumoniae), are the major culprits. N. meningitidis is responsible foran estimated 24-25% of meningitis in children one month of age through15 years; for adults, the figure is 10-35%. H. influenzae is responsiblefor an estimated 40-60% of meningitis cases in children one month of agethrough 15 years, while S. pneumoniae is responsible for 10-20% ofmeningitis cases in the same age group, as well as 30-50% of cases inadults (over 15 years). [W. K. Joklik et al. (eds.), ZinsserMicrobiology, 18th ed., p. 485, Appleton-Century-Crofts, Norwalk, Conn.(1984).] Other organisms such as Streptococcus spp. in groups A and B,Staphylococcus aureus, Listeria monocytogenes, and various gram-negativebacilli (e.g., enterics such as E. coli) are responsible for sporadiccases. Untreated, bacterial meningitis is fatal in 70-100% of patients,and infected neonates may have motor or intellectual impairment relatedto their infection. [J. M. Slack and I. S. Snyder, Bacteria and HumanDisease, pp. 128-133, Yearbook Medical Publishers (1978).]

The blood-brain barrier represents a significant obstacle to treatmentof meningitis, especially prophylactically. As the barrier is designedto prevent invasion of organisms and uptake of compounds (e.g.,antimicrobials), intravenous anti-microbial administration is not alwayssufficient. For example, estimates provided in experimental studiesindicate that drug concentrations in the cerebrospinal fluid and brainare approximately 1/200 to 1/500 of those in serum. [G. P. Youmans etal., Biologic and Clinical Basis of Infectious Diseases, 3d ed., p. 553,W. B. Saunders Co., (1985).] Even with the inflammatory changesassociated with an intensity characteristic of bacterial meningitis,passage of antimicrobials is hindered by the barrier. [Id.]

Endotoxemia due to the release of endotoxins from dividing organisms andthe presence of endotoxin in the cerebrospinal fluid (CSF) presentserious complications during sepsis and meningitis. Endotoxin isdetectable in the plasma and CSF of patients with meningitis due togram-negative bacteria. (Awad et-al., supra at 560.) Perhaps due toincreased permeability of the bowel mucosa, endotoxin may also be foundin the plasma of patients with meningitis due to gram-positive organisms(e.g., Streptococcus pneumoniae).

Ironically, release of endotoxin is aggravated by antimicrobialtreatment. Indeed, it is believed that aggressive antibiotic treatmentcan be life-threatening. This is due to the increased burden ofendotoxin present in the blood and CSF which results when a large numberof organisms are simultaneously killed by the antibiotic. This increasedendotoxin burden results in the pathology associated with fatalmeningitis and is a significant problem facing clinicians who must treata seriously ill patient within the first few hours of disease.

Therefore, the present invention contemplates treating acute septicconditions with anti-C5a antibodies. It is contemplated that theseantibodies be administered alone, or in combination with othertherapeutic preparations. In preferred embodiments, the presentinvention provides a method comprising; providing; a subject sufferingfrom symptoms of sepsis, a therapeutic composition comprising anantibody specific for complement component C5a peptide, andadministering the therapeutic composition to the subject.

C. Veterinary Care

Septicemia and sepsis are by no means limited to human beings. Infectionby gram-negative bacteria accounts for significant morbidity andmortality in neonatal livestock, such as calves. [D. D. Morris et al.,Am. J Vet. Res. 47:2554-2565 (1986).] Interestingly, humoral immunestatus is again related to susceptibility to sepsis and this is largelydependent on passive transfer from colostrum. For this reason, in someembodiments the present invention contemplates determining the immunestatus of the animal prior to administration of the anti-C5a antibodies.This determination can be made by screening neonatal calves for totalcirculating serum immunoglobulin (e.g., by ELISA).

Where the immune status is poor (e.g., low total IgG levels), theantibodies of the present invention should be used prophylactically.Where the animal's immune status is healthy, use of the antibodies maybe needed for acute therapy of gram-negative bacterial sepsis, whichremains prevalent in neonatal calves even with high natural antibodylevels.

The present invention contemplates the treatment of other animals aswell. For example, among foals less than 10 days of age in criticaldistress, sepsis is the most serious problem. [A. M. Hoffman et al., J.Vet. Int. Med. 6:89-95 (1992).] Symptoms highly indicative of sepsisrisk include weakness, metabolic disturbance and dehydration. In oneembodiment, the invention contemplates using antibodies for prophylactictreatment of foals less than 10 days of age having these indicators, orthose at risk of infection.

While positive blood cultures are found in less than half of the cases,those animals found positive have a very poor chance of survival. Thepresent invention therefore contemplates using anti-C5a antibodies foracute treatment of any animal with evidence of septicemia, with orwithout culture-proven cases.

IV. Therapeutic Preparations And Combinations

In some embodiments, the present invention contemplates usingtherapeutic compositions of soluble anti-C5a antibodies. It is notintended that the present invention be limited by the particular natureof the therapeutic composition. For example, such compositions can beprovided together with physiologically tolerable liquids, gels, solidcarriers, diluents, adjuvants and excipients (and combinations thereof).In addition, anti-C5a antibodies may be used together with othertherapeutic agents, including other immunoglobulins or antibiotics.

As noted above, these therapeutic compositions can be administered tomammals for veterinary use, such as with domestic animals, and clinicaluse in humans in a manner similar to other therapeutic agents. Ingeneral, the dosage required for therapeutic efficacy varies accordingto the type of use and mode of administration, as well as theparticularized requirements of individual hosts. The attending medicalprofessional is capable of determining the therapeutically effectivedosage based on the characteristics of the subject (e.g. gender, age,weight, etc.)

With respect to the mode of administration, in some embodiments theantibodies are administered intravenously, intramuscularly,subcutaneously, intradermally, intraperitoneally, intrapleurally,intrathecally, or topically. In some embodiments, formulations for suchadministrations may comprise an effective amount of anti-C5a antibodiesin sterile water or physiological saline.

On the other hand, formulations may contain such normally employedadditives as binders, fillers, carriers, preservatives, stabilizingagents, emulsifiers, buffers and excipients as, for example,pharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharin, cellulose, magnesium carbonate, and the like. Thesecompositions typically contain 1%-95% of active ingredient, preferably2%-70%.

The compositions are preferably prepared as injectables, either asliquid solutions or suspensions; solid forms suitable for solution in,or suspension in, liquid prior to injection may also be prepared.

The antibodies of the present invention are often mixed with diluents orexcipients which are compatible and physiologically tolerable. Suitablediluents and excipients are, for example, water, saline, dextrose,glycerol, or the like, and combinations thereof. In addition, ifdesired, the compositions may contain minor amounts of auxiliarysubstances such as wetting or emulsifying agents, stabilizing or pHbuffering agents.

Where repeated administrations are required, it may be beneficial tofirst clear any anti-hapten antibodies by administering free antibiotic.This can then be followed by administration of the anti-C5a antibodiesof the present invention.

Experimental

The following examples are provided in order to demonstrate and furtherillustrate certain preferred embodiments and aspects of the presentinvention and are not to be construed as limiting the scope thereof.

In the experimental disclosure which follows, the followingabbreviations apply: N (normal); M (molar); mM (millimolar); μM(micromolar); mol (moles); mmol (millimoles); μmol (micromoles); nmol(nanomoles); pmol (picomoles); g (grams); mg (milligrams); μg(micrograms); ng (nanograms); 1 or L (liters); ml (milliliters); μl(microliters); cm (centimeters); mm (millimeters); μm (micrometers); run(nanometers); °C. (degrees Centigrade); Sigma (Sigma Chemical Co., St.Louis, Mo.).

EXAMPLE 1 Induction of Sepsis In Rats by Cecal Ligation Puncture

This Examples describes the induction of sepsis by Cecal LigationPuncture (CLP) in Male Long-Evans specific pathogen-free rats (275-300mg) obtained from Harlan, Inc., Indianapolis, Ind. Anesthesia wasinduced by intraperitoneal administration of ketamine (20 mg/100 mg bodyweight). Through a 2 cm abdominal midline incision the cecum was ligatedbelow the ileocecal valve without obstructing the ileum or colon. Thececum was then subjected to a single “through and through” perforationwith a 21-gauge needle. After repositioning the bowel, the abdominalincision was closed in lagers with plain gut surgical suture 4-0(Ethicon, Inc., Somerville, N.J.) and metallic clips. Sham animalsunderwent the same procedure except for ligation and puncture of thececum.

EXAMPLE 2 Preparation and Characterization of Anti-rat C5a Antibodies

This Example describes the preparation and characterization of anti-ratC5a antibodies. Rat C5a peptide with the sequence KHRVPKKCCYDGARENKYET(SEQ ID NO:2) was obtained from Research Genetics (Huntsville Ala.) andcoupled to keyhole limpet hemocyanin (KLH). This 20-mer rat C-terminaltruncated C5a peptide corresponds to amino acid residues 17-36 of thefull length rat C5a peptide [Rothermel, E. et al., Biochimica. etBiophysica. Acta. 1351:9-12, (1997)], which is listed as SEQ ID NO: 1.The coupled peptide was used as an antigen to immunize rabbits. Afterseveral injections, the antibody was affinity purified using thesynthetic 20-mer peptide coupled to beads. Immunoprecipitation withactivated rat serum using beads coupled with this antibody yielded asingle band with a 14 kDa position in Western blot analysis,characteristic of rat C5a peptide [See, Ward, P. A. and Becker, E. L.,J. Exp. Med. 127:693-709 (1968)].

When added at a concentration of 100 μg/ml, the antibody did not affectwhole hemolytic complement activity (CH50) of fresh rat serum. Additionof 0, 10, 20, 40, 80 micrograms of anti-C5a Ab yielded CH50 values of99, 94, 91, 93, and 94 units/ml. Therefore, the fact that CH50 levelsare not affected by the anti-C5a antibodies indicates that the anti-C5aantibodies are effective without compromising the entire complementsystem (including the classical and alternate pathways).

EXAMPLE 3 Anti-Rat C5a Antibodies Prevent Sepsis

This Example describes the treatment of sepsis. CLP-induced sepsis wasgenerated in three different groups of rats according to the procedureof Example 1 above. The first group (n=21) was treated intravenouslywith 400 μg of preimmune IgG (in a volume of 300 μg) immediately afterthe CLP procedure. The second group (n=10) was treated intravenouslywith 400 μg of anti-C5a IgG prepared according to Example 2 above (in avolume of 300 μg) immediately after the CLP procedure. The third group(n=12) was depleted of C3 by four intraperitoneal injections of purifiedcobra venom factor (25 units per injection at 12 hour intervals) duringthe 48 hour period prior to induction of CLP. This protocol has beenshown to reduce serum C3 levels to less than 3% of normal and tosuppress whole complement hemolytic activity (CH50) to undetectablelevels [Hill, J. H. and Ward, P.A., J. Exp. Med. 133:885-900, (1971)].

Survival rates for the three groups of rats were determined over aten-day period, with assessment every 6 hours. A sham operated group ofrats (n=10) in which no cecal puncture was performed, were also includedin this study and all survived during the 10 day period.

All animals had unlimited access to food and water, both pre- andpostoperatively. Survival data for the three CLP treated groups isdepicted in FIG. 1. In the CLP group receiving preimmune IgG, survivalwas 66.7% (14/21) 24 hours after CLP, diminishing progressively each dayuntil day 8, at which time only 9.5% (2/21) were alive. In the C3depleted group, the survival time was greatly reduced, with all animalsdead before day 4.5. When this group was compared to the CLP groupreceiving preimmune IgG, the outcomes were statistically different(p=0.01 by the Chi square test). In the CLP group receiving anti-C5aantibodies, survival times were dramatically improved. By day 5, 50% ofthe animals survived, and no additional deaths were observed in the next5 days. When the CLP group receiving preimmune IgG was compared to thegroup receiving anti-C5a IgG, the p values for 10 day survival were0.012 and 0.022 by Chi Square Test and Fisher's exact test respectively.Comparing the outcome of the CLP group receiving anti-C5a antibodies tothe CLP group depleted of C3, p equaled 0.01 according to the Chi squaretest.

EXAMPLE 4 Anti-Rat C5a Antibodies Reduce Bacteremia in Septic Rats

This Example describes the in vitro detection and reduction ofbacteremia in septic rats. CLP-induced sepsis was generated in twodifferent groups of rats according to the procedure of Example 1 above.One group (n=5) was treated intravenously with 400 μg of preimmune IgG,which was infused immediately after surgery. The second group (n=5) wastreated intravenously with 400 μg of anti-C5a IgG (prepared according toExample 2), which was infused immediately after surgery. A third groupof sham operated rats (n=5) in which no cecal puncture was performed,were also included.

CLP-induced sepsis in rats is known to be associated with thedevelopment of bacteremia involving the presence of both aerobic andanaerobic bacteria in the blood [Deitch, E. A. Schock 9:1-11, (1997)].Blood samples were obtained from these three groups 36 hours after shamsurgery or induction of CLP. This was done by drawing blood via theposterior vena cava after topical treatment of the puncture site withiodine swabsticks (Professional Disposables, Inc., Orangeburg, N.Y.).Blood samples were placed in Isolator Microbial Tubes (Wampole, Inc.)and cultured on chocolate sheep blood agar plates (incubated aerobicallyin 5% CO₂) or on lysed blood agar plates (incubated anaerobically). Allplates were incubated for 96 hours prior to determination of colonycounts (cfu). The presence of aerobic and anaerobic bacteria in bloodwas measured as cfu is shown in FIG. 2.

In the sham operated group at 36 hours, cfu were not detectable. In theCLP group receiving preimmune IgG, the cfu value (x±SEM) was 740±328,while in the CLP group treated with anti-C5a antibodies, the cfu valuewas profoundly reduced, by 98% (p<0.05), to 18±10 cfu. When subculturesof mixed aerobic or anaerobic bacteria obtained from the blood of CLPrats incubated in the presence of either preimmune IgG or anti-C5a IgG(each at 100 μg/ml), no reduction in cfu values were found, indicatingthat anti-C5a antibodies are not directly bacteriostatic.

Bacteremia was also assayed in the organs of both sham and CLP treatedrats, which received either preimmune IgG or anti-C5a antibodies(prepared according to Example 2). Livers and spleens were obtained fromthese rats 36 hours after surgery. These tissues were homogenized andcfu/g of tissue was determined using procedures similar to those usedfor the blood samples described above. The data is shown in FIG. 3, inwhich the cfu values for aerobic and anaerobic bacteria were arbitrarilyaggregated. In all cases, there was a 1:1 ratio of these two classes ofbacteria. In sham animals and in CLP animals treated with anti-C5a IgG,the cfu values were low in both liver and spleen. Between these twogroups and in both organs, there were no statistically significantdifferences in cfu values. In marked contrast, CLP rats treated withpreimmune IgG had very high cfu values (3-11×10⁶/g tissue).

EXAMPLE 5 Anti-Rat C5a Antibody Reduces C5a Binding to Neutrophils

This Example describes the binding of C5a peptide to neutrophils in theblood of septic rats, and ability of anti-rat C5a antibodies to reducethis binding. Blood neutrophils were obtained from both sham and CLPtreated rats, which received either preimmune IgG or anti-C5a antibodies(prepared according to Example 2) at 12, 24 and 36 hours after CLP.Neutrophils were evaluated in flow cytometry for the surface content ofC5a peptide using the procedure described below.

Whole blood was recovered from these rats, being drawn into syringescontaining the anticoagulant ACD (Baxter Health Care Corp., Deerfield,Ill.). Duplicate aliquots (250 μl) of cells were incubated with an equalvolume of phosphate buffered saline. The phosphate buffer, pH 7.4, wasmade up with heat inactivated 1% fetal bovine serum and 0.1% NaN₃,containing 10 μg/ml of either anti-rat C5a polyclonal antibody (as usedin Mulligan, M. S. et al., J. Clin. Invest. 98:503-512, 1996) orirrelevant rabbit IgG control antibody (Jackson Laboratories, BanHarbor, Me.). The cells were incubated at 5° C. for 30 minutes. Cellswere washed once and red blood cells lysed with FACS solution (BectonDickinson, San Jose, Calif.). Cells were then washed and incubated withphycoerythrin labeled anti-rabbit IgG (Sigma Chemical Co., St. Louis,Mo.). Cells were washed twice and suspended in 400 μl PBS containing 2%paraformaldehyde. Phycoerythrin intensity of gated populations(identified as forward versus side scatter light) was measured on aFACScan Flow Cytometry System (Becton Dickinson) in which 10,000 cellsper gate were counted and the amount of phycoerythrin analyzed usingPC-LYSYS software (Becton Dickson).

The data collected from this procedure is shown in Table 3. Mean channelfluorescence (MCF) values for neutrophils obtained from sham operatedrats or from CLP rats at 12 hours showed very low MCF values for C5apeptide. By 24 and 48 hours, neutrophils from CLP animals showed nearly4 fold increases in binding of anti-C5a IgG, suggesting that these cellscontained substantial amounts of C5a on their surfaces. Binding ofpreirnmune IgG to neutrophils from CLP rats at 24 and 36 hours was verylow (less than 10 MCF units). Thus, during CLP induced sepsis, bloodneutrophils acquire C5a peptides on their surfaces. When C5a peptidecontent was evaluated on blood neutrophils obtained from CLP ratspre-treated with 400 μg anti-C5a, there were consistent reductions inC5a content at 24 and 36 hours when compared to neutrophils from CLPrats pre-treated with preimmune IgG.

TABLE 4 Detection by Flow Cytometry of C5a Peptides on Blood NeutrophilsDuring Sepsis Mean Channel Fluorescence (mean ± SEM) Group 0 hour 12hour 24 hour 36 hour Sham 12.8 ± 1.48* CLP + 11.9 ± 0.54 43.5 ± 1.4245.6 ± 0.64 preimmune IgG (400 μg) CLP + 10.6 ± 0.37 32.6 ± 0.34 32.3 ±2.74 anti-C5a IgG (400 μg) *All displayed binding values representresults with rabbit anti-C5a IgG as detected with phycoerythrin labeledanti-rabbit IgG. The binding value of preimmune IgG was 4.45 ± 0.16. Foreach data point, n = 4 and all samples were analyzed in quadruplicate.

EXAMPLE 6 Anti-rat C5a Antibody Preserves H₂O₂ Production in Septic RatNeutrophils

This Example describes the ability of anti-rat C5a antibody to preservethe H₂O₂ production of neutrophils from septic rats. Blood neutrophilswere obtained from both sham and CLP treated rats, which received 400 μgof either preinunune IgG or anti-C5a antibodies (prepared according toExample 2) 36 hours after CLP or sham surgery. Neutrophil generation ofH₂O₂ was assayed using the procedure described below.

Neutrophils were isolated from blood using dextran sedimentation andhypotonic red blood cell lysis. 7.5×10⁵ cells were suspended in Hank'sbalanced salt solution (in a final volume of 1.0 ml) in the presence orabsence of catalase (100 units/ml) in a final volume of 1.0 ml.Neutrophils were then stimulated for 1 hour at 37° with phorbolmyristate acetate (PMA) at a concentration of 100 ng/ml. Stimulation wasterminated by addition of 10% (vol/vol) trichloroacetic acid. Afterremoving precipitated protein by centrifugation (10 minutes at 500× g),10 mM ferrous ammonium sulfate (0.2 ml) and 2.5 M potassium thiocyanate(0.1 ml) were added to the sample. The presence of the ferrithiocyanatecomplex formed in the presence of peroxide was measured at 480 nm andcompared to a standard curve generated using dilutions of stock H₂O₂.

The data collected from this procedure is shown in FIG. 4. Very littleH₂O₂ (circa 0.1 nmol) was produced in unstimulated blood neutrophilsobtained from sham rats or from CLP rats pre-treated with preimmune IgGor anti-C5a IgG. After PMA stimulation, neutrophils from sham ratsproduced 3.1±0.75 nmol H₂O₂. In CLP rats treated with preimmune IgG,H₂O₂ production of PMA-stimulated neutrophils was reduced by nearly 62%,to 1.25±0.50 nmol. In striking contrast, blood neutrophils from CLPanimals treated with anti-C5a demonstrated full H₂O₂ generation,3.58±0.67 nmol after in vitro stimulation with PMA, indicating thattreatment with anti-C5a antibodies preserved this response inneutrophils from CLP rats.

EXAMPLE 7 C5a Peptides Reduce Chemotaxis of Human Neutrophils

This Example describes the ability of certain synthetic peptidesrepresentative of regions of human C5a peptide to reduce the chemotacticresponse of human neutrophils to human C5a peptide. Human neutrophilswere isolated from human blood by traditional Ficoll-Hypaquesedimentation techniques. Using standardized methodology, neutrophils(5×10⁶/ml) labeled with 1 μg/ml BCECF [2′,7′-(2carboxyethyl)-5-(and-6)-carboxyfluroscein, acetoxymethy ester) at 37° C.for 30 min, were applied to the upper compartments of chemotacticchambers and evaluated for their motility responses to 10 nM human C5apeptide (SEQ ID NO:3) added to the lower compartments. The twocompartments were separated by a membrane with pore sizes of 3 μm.Neutrophil chemotactic responses were quantitated by cytofluorometry.

Three different peptides, labelled A, M, and C, were also addedseparately to the lower compartments, at a concentration of 1 μM, inmedium alone or together with 10 nM human recombinant C5a peptide.Peptide A (SEQ ID NO:4) represents residues 1-20 of human C5a peptide,peptide M (SEQ ID NO: 5) represents residues 21-40 of human C5a peptide,and peptide C (SEQ ID NO:6) represents residues 55-74 of human C5apeptide. As shown in FIG. 5, the presence of M from regions A, M, or C,did not per se induce any chemotactic responses. However, the presenceof 1 μM of any of these three peptides with 10 μM human C5a resulted insignificant reduction (approximately 20%) in the chemotactic responsesof neutrophils. This data suggests that peptides from the N-terminal,mid-portion, and C-terminal regions of human C5a have the ability tocompete functionally with intact C5a peptide, while demonstrating nointrinsic chemotactic activity.

EXAMPLE 8 Neutrophil Chemotactic Activity of C5a Peptides Linked to KLH

This Example describes neutrophil chemotactic activity of peptides A, M,and C linked to keyholelimpet hemocyanin (KLH) employing the chemotacticassay described in Example 7. The peptide:KLH molar ratios employed wereapproximately 3:1. The chemotactic responses of neutrophils wasevaluated to medium alone, to 10 nM recombinant C5a peptide (SEQ IDNO:3), and to the KLH-A, M, or C conjugates (SEQ ID NOS:4, 5, and 6respectively), at the calculated synthetic peptide concentrations of 100nM.

The data collected in this Example is shown in FIG. 6. The A peptide KLHconjugate was the most chemotactically active compound when compared toC5a peptide, while the C peptide conjugate was almost as active. The Mpeptide conjugate revealed no chemotactic activity.

EXAMPLE 9 Polyclonal Rabbit Anti-Human C5a Reactivity with Human C5aPeptide

In this Example epitopes in human C5a peptide were evaluated forreactivity with commercially available rabbit polyclonal anti-human C5aantibodies (purchased from Calbiochem-Novabiochem Corp., San Diego,Calif.). Thirty-four (34) μg of this polyclonal anti-human C5a IgG wasincubated with 20 μg KLH peptide conjugates (A, M, C, as described inExample 8) for 18 hours at 4° C. Treated and untreated antibodies werethen evaluated for their ability to react with recombinant human C5apeptide by Western blot analysis. Fifty (50) ng C5a peptide was added toeach lane, and electrophoresis was carried out. As shown in FIG. 7, twobanding patterns (one between the 6.5 and 14.3 kDa markers and the othernear the 21.4 kDa marker) were found by Western blot analysis. Whenanti-C5a antibody was incubated with human recombinant C5a peptide, bothbands disappeared in Western blots. Preabsorption of the antibody with Cpeptide abolished the slower band and nearly abolished the fastermigrating band. Absorption with M peptide greatly diminished theintensity of both bands, while preabsorption with A peptide showedlittle evidence of removal of reactivity of the antibody with C5apeptide. This data suggests that the commercially available polyclonalrabbit antibody to human C5a peptide is most reactive with the Cterminal region of C5a peptide (represented by peptide C), less reactivewith the mid-region of C5a (represented by peptide M), and little, if atall, reactive with the N terminal region of human C5a (represented bypeptide A).

EXAMPLE 10 Peptides Which Antagonize the Binding of Human C5a Peptide toNeutrophils

This Examples describes certain peptides which are able to antagonizethe ability of human C5a peptide to bind to human neutrophils. Threedifferent peptides, labelled A, M, and C, were used to antagonize thebinding of ¹²⁵I-hC5a peptide to human neutrophils. Again, peptide A (SEQID NO:4) represents residues 1-20 of human C5a peptide, peptide M (SEQID NO: 5) represents residues 21-40 of human C5a peptide, and peptide C(SEQ ID NO:6) represents residues 55-74 of human C5a peptide.

Human peripheral blood neutrophils (1×10⁷ cells/ml) were incubated inHank's buffered saline plus 0.1% bovine serum albumin with both¹²⁵I-labelled hC5a peptide (300μCi/nmol) and either peptide A, M, or C,in a final volume of 200 μl in a microfuge tube. The ratio of theshorter peptides (A, M, or C) to ¹²⁵I-labelled hC5a peptide was 10:1.After incubation, cell suspension were layered over a 20% sucrosegradient and were sedimented by centrifugation at 11,000 g. The tubeswere then frozen on dry ice, followed by cutting the tips containing thepellet. Cell-bound label was then determined by placing the tips in agamma counter.

As shown in FIG. 9, peptides A and M were significantly (p<0.05)competitive in reducing the binding of hC5a peptide, whereas the Cterminal peptide (peptide C) showed no statistically significantinterference.

EXAMPLE 11 Screening Candidate Peptides for Useful Immunogens

This Example describes three methods which are employed in screeningcandidate C-terminal truncated C5a peptides for useful immunogens (i.e.which can be used to produce the anti-C5a antibodies of the presentinvention). One method involves screening C-terminal truncated C5apeptides which inhibit the chemotaxis of neutrophils. Another methodinvolves screening peptides for the ability to antagonize the binding ofC5a peptides to neutrophils. A third method involves administeringcandidate C-terminal truncated C5a peptides to septic animals andmonitoring their response.

The first method, as described in Examples 7 and 8, is used to screencandidate C-terminal truncated C5a peptides which inhibit the chemotaxisof neutrophils. With this method, human neutrophils are isolated fromhuman blood by traditional Ficoll-Hypaque sedimentation techniques.Using standardized methodology, neutrophils are applied to the uppercompartments of chemotactic chambers and evaluated for their motilityresponses to 10 nM human C5a peptide (SEQ ID NO:3) added to the lowercompartments. The two compartments are separated by a membrane with poresizes of 3 μm. Neutrophil chemotactic responses are quantitated bycytofluorometry.

A candidate C-terminal truncated peptide (1 μM), which may be linked toKLH, is added to the lower compartments in medium alone or together with10 nM human recombinant C5a peptide. The chemotactic response of theneutrophils is then quantitated by cytofluorometry. One indication of auseful immunogen is if the candidate C-terminal truncated C5a peptideinduces chemotaxis of the neutrophils as compared to human C5a peptidealone. Another indication of a useful immunogen is if the candidateC-terminal truncated C5a peptide inhibits chemotaxis of neutrophils whencombined with human C5a peptide, as compared to human C5a peptide alone.

A second method, as described in Example 10, is used to screen candidateC-terminal truncated C5a peptides which antagonize the binding of¹²⁵I-C5a peptide to neutrophils. In this Example, human neutrophils areincubated with human ¹²⁵I-C5a peptide and the candidate C-terminaltruncated C5a peptide. Inhibition of ¹²⁵I-C5a peptide binding to theneutrophils by the candidate C-terminal truncated peptide indicates apotentially useful immunogen for the production of anti-C5a antibodies.

A third method employs the septic rats described in Example 1. In thisExample, one group of CLP sepsis induced rats is administered 50 mg/kgof a candidate C-terminal C5a truncated peptide intravenouslyimmediately after the CLP procedure, while a second group of CLP rats isused as a control. Survival rates and symptoms of sepsis are recordedover a ten day period. Candidate C-terminal truncated C5a peptides whichreduce the symptoms of sepsis, and/or increase survival times ascompared to the control group are considered potential immunogens forproducing anti-C5a antibodies.

EXAMPLE 12 H₂O₂ Production of Human Neutrophils In The Presence of ShortC5a Peptides

This Example describes the effect of human C5a peptide, and certainshorter synthetic human C5a peptides A, M, or C, to inhibit the H₂O₂production of human neutrophils stimulated by phorbol myristate acetate(PMA). Again, peptide A (SEQ ID NO:4) represents residues 1-20 of humanC5a peptide, peptide M (SEQ ID NO: 5) represents residues 21-40 of humanC5a peptide, and peptide C (SEQ ID NO:6) represents residues 55-74 ofhuman C5a peptide. Human neutrophils were isolated and then pretreatedwith C5a peptide or one of the shorter synthetic peptides (A, M, or C).Cells were then stimulated with PMA and the production of H₂O₂ wasmeasured.

As shown in FIG. 10, C5a peptide and the shorter peptides alone have noeffect on H₂O₂ production. However, PMA elicits a strong H₂O₂ response,which is inhibitable by the C5a peptide, and to varying degrees bypeptides A, M, and C. Peptide M was shown to exert the strongestsuppression of H₂O₂ production of all of the peptides (See FIG. 10).

EXAMPLE 13 Preparation and Characterization of Anti-human C5a Antibodies

This Example describes the production of anti-human C5a antibodies. Ashort human C5a peptide with the sequence CCYDGASVNNDETCEQRAAR (peptideM, SEQ ID NO:5) was obtained from Research Genetics (Huntsville Ala.)and coupled to keyhole limpet hemocyanin (KLH). The coupled peptide wasused as an antigen to immunize rabbits. After several injections, theantibody was recovered by affinity purification.

A Western Blot was then used to demonstrate that these anti-human C5aantibodies are specific for human C5a peptide, and not rat C5a peptide.Briefly, recombinant human and rat C5a peptide were run in parallel on agel, and transferred to a filter. The filter was blocked, and thenprobed with the anti-human C5a antibody. The resulting signal revealedthat the anti-human C5a antibody was only able to recongnize human C5apeptide, and not rat C5a peptide (See FIG. 11).

An additional Western blot was also performed in order to determine ifpolyclonal anit-human antibodies (commercially available fromCalbiochem) shared this type of specificity. Again, recombinant humanand rat C5a peptide were run in parallel on a gel, and transferred to afilter. The filter was blocked, and then probed with the commerciallyavailable polyclonal anti-C5a antibody. The resulting signal revealedthat the polyclonal anti-human C5a antibody was able to recognize bothhuman and rat C5a peptide (See FIG. 11). As such, it is clear that thecommercially available polyclonal anti-human C5a antibody does not havethe same type of specificity as the anti-human C5a antibody discussedabove.

EXAMPLE 14 Therapeutic Use of Anti-Human C5a Antibodies to Treat Sepsisin Humans

Anti-C5a antibodies may be used prophylactically or therapeutically totreat sepsis in humans. Individuals at risk of contracting sepsis,particularly patients undergoing surgery, or those with sepsis may beadministered an effective amount of anti-C5a antibodies to prevent orreduce the severity of the disease. A typical treatment regimen wouldconsist of administering 5-10 mg of antibody per kilogram of patientbody weight. Prophylactically the dose would be given just prior tosurgery, and repeated at least once immediately thereafter.Therapeutically the dose would be given every 24-48 hours untilremission of the disease is apparent. The initial therapeutic dose wouldbe 25 mg per kilogram of patient body weight, and then reduced to 5-10mg per kilogram. The antibody may be administered by any number ofroutes, but the preferred route of administration is intravenously.

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention which are obvious to those skilled inbiochemistry, immunology, chemistry, molecular biology, the medicalprofession or related fields are intended to be within the scope of thefollowing claims.

1. A composition comprising an isolated and purified peptide consistingof an amino acid sequence selected from the group consisting of SEQ IDNO: 14, SEQ ID NO: 15, and a portion thereof, wherein said portion isfive amino acids in length.
 2. The composition of claim 1, wherein saidpeptide is suitable for the reduction of C5a binding to neutrophils. 3.The composition of claim 1, further comprising adjuvant.
 4. Thecomposition of claim 3, wherein said adjuvant is selected from the groupconsisting of agar beads, aluminum hydroxide, aluminum phosphate,incomplete Freund's, Quil A and bacterin.